Over the last three decades, cervical cancer (CC) control has been based on screening and treatment of higher grades of cervical intraepithelial neoplasia (CIN 2+). This has been the standard of care despite the fact that only a subset of such lesions may progress to CC if left untreated and that in younger women a substantial proportion of CIN 2 may spontaneously regress. Treatment of lesions which are likely to regress spontaneously is likely unnecessary, but the fact that more than 30% of CIN 2 in younger women (< 25 years old) may progress to CIN 3+ cannot be ignored specially in populations where the compliance with follow-up is poor. This project seeks to examine whether cervical microbiome profiles/associated biomarkers can be used to identify CIN 2 of younger women with molecular features of CIN 2 or 3 of older women (e 25 years old) that are treated under the current guidelines. If successful, this approach could be utilized to prevent overtreatment of younger women with CIN 2 and thus ensure no adverse reproductive or obstetrical outcomes occur while making sure those at highest risk of progression are afforded appropriate CC prevention treatment. It is increasingly recognized that microbes in human body sites play major roles in modifying several human diseases including cancer. Our preliminary studies, which employed genetic sequencing of 16S ribosomal RNA genes demonstrated the ability to generate microbial profiles using cervical mucus (CM) samples of women diagnosed with abnormal pap. Since younger women are more likely to have an adverse microbiome, we propose to take advantage of this novel technology to investigate differences in cervical microbiome among younger women with CIN 2 and older women with CIN d1, CIN 2 or 3. Altered cervical microbiome/dysbacteriosis and associated inflammation cause oxidative stress and may result in adverse alterations in DNA (damage, instability and alterations in global DNA methylation) and facilitate HPV integration, a necessary event in the process of CC. Therefore, it is conceivable that in addition to altered cervical microbiome profiles, changes in DNA markers related to microbiome-induced oxidative stress or HPV integration, namely, 8-hydroxy-2'-deoxyguanosine [8-OHdG], telomere length (TL), global DNA methylation and p16INK4A will be able to distinguish severe CIN 2 that are likely to progress to CIN 3+ from less severe CIN 2 that are likely to regress in younger women by comparing the molecular features of those with CIN 2/3 of older women. Following are the specific aims. Aim 1: Examine differences and similarities in microbial profiles of CIN 2 of younger women with CIN d1, CIN 2, or CIN 3 of older women, (n=125 each of 4 CIN age groups; N=500). This aim will be tested using DNA extracted from CM samples from 500 women. Aim 2: Evaluate whether microbial profiles identified in Aim 1 are associated with oxidative stress/HPV integration markers, i.e., 8-OHdG and p16INK4A in paraffin embedded cervical specimens and TL and the degree of DNA methylation in long interspersed nucleotide element-1 (L1) in exfoliated cervical cell DNA of women stated in Specific Aim 1.